Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

SOME PHYTOCHEMICAL AND ENZYMATIC STUDIES ON SEEDS

OF WILD OATS AND COMMON LAMBSQUARTERS ASSOCIATED

WITH WHEAT CROP

Asma Saeed1,*, Muhammad Salim Marwat1, Rubina Naz2, Asif Latif

Baloch3, Jaweria Gul4, Inayatullah Awan5 and Ahmad Saeed6

ABSTRACT

Acid phosphatases (EC 3.1.3.2) catalyze the hydrolysis of

various phosphomonoesters at pH below 6.0. These enzymes from two

sources, Avena fatua (wild oats) and Chnopodium album (common

lambsquarters) seeds were partially purified by chromatography on

DEAE-Cellulose and Sephadex G-100 columns to make an appropriate

comparison of their properties and characteristics. Both enzymes had

obtained specific activity in range of 460-1000 units/mg of protein with

approximately 20-40 fold purification and had overall recovery of

about 60 %. Both enzymes were found fully stable at pH 5-6 and 20-

37o C. Both enzymes showed pH optima of 5.6, but the temperature

optima of 55o C for C. album seeds and 60o C for A. factua seeds were

obtained. The Km of enzymes against substrate, p.nitrophenyl

phosphate was 0.3mM and 0.23mM, respectively. Substrate specificity

indicated that p.nitrophenyl phosphate, phenyl phosphate, ATP and

sodium pyrophosphate were found good substrates. Kinetic

parameters suggested that the enzyme from C. album was more

active than that of A. fatua. Phytochemical analysis of weed seed

extracts was carried out. Alkaloids, saponins, glycosides, terpenoids,

steroids, flavonoids and tannins were detected. Tannins and flavinoids

were in high concentration while alkaloids and saponins were present

in low concentrations. Flavonoids are water soluble polyphenolic

molecules thought to provide antioxidant effects and these plants may

be used to protect oxidative damage of biomolecules in human body.

Key words: Acid phosphatases, alkaloids and tennins, Avena fatua,

characterization, Chenopodium album, Purification.

Citation: Saeed, A., M.S. Marwat, R. Naz, A.L. Baloch, J. Gul, I. Awan

and A. Saeed. 2016. Some phytochemical and enzymatic studies on

1Dept of Biological Sciences, 2Dept. of Chemistry, 3Dept. of

Horticulture, 5Dept. of Agronomy, Gomal University, DI Khan, Pakistan 4Dept. of Biotechnology, SBB University, Sheringal, Upper Dir, Pakistan 6Dept. of Chemistry, Qurtuba University of Science and Information

Technology, Dera Ismail Khan 29050, Pakistan

*Corresponding author’s email: asmasaeed78@gmail.com

Asma Saeed et al., Some phytochemical and enzymatic ... 212

seeds of wild oats and common lambsquarters associated with wheat

crop. Pak. J. Weed Sci. Res. 22(2): 211-226.

INTRODUCTION

There are more than 30 weed species in the wheat fields of

Pakistan (Qureshi and Bhatti, 2001; Siddiqui and Bajwa, 2001).

Among these, Avena fatua and Chenopodium album are found to be

the most frequently occurring weeds. They reduce the wheat grain

yield by 30-60% in the fields. There is a dire need of herbicides to

control the weeds of wheat (Bibi et al., 2008; Cheema et al., 2006;

Usman et al., 2010). Other species like Phalaris minor, Vicia sativa,

Galium aparine, Rumex dentatus, Lathyrus aphaca, Carthamus

oxycantha, Convolvulus arvensis and Euphorbia sp. in association with

wheat crop plant have also ethno-botanical importance. These weeds

have been extensively used in herbal medicine and also offering

feeding, sheltering, refuge and breeding niches to beneficial

arthropods. Their seeds dormancy and irregular germination

throughout the growing season are the most important features

contributing to the persistence of weeds.

Avena fatua is very dangerous weed grown in wheat, oat and

barley crops which lowers the quality of these crops through

competing for their resources. Various diseases and pests such as fruit

fly and smut are associated with this self growing weed. A. fatua seeds

are nerve tonic, stimulant and laxative (Hussain et al., 2006).

Chenopodium album grows very fast in wheat fields. It is

commonly used for food and medicinal values (Yadav et al., 2007). Its

leaves are cooked like spinach. Its black seeds are rich in proteins,

alkali and alkaline earth metal ions together with some vitamins. It is

used for cough and abdominal pain. Chenopodium album has

antipruritic and antinociceptic activities (Yadav et al., 2007). The

literature survey (Abbas et al., 2012) revealed that varieties of natural

products are present in seeds of two plants namely C. album (dicot)

and A. fatua (monocot). These secondary metabolites act as natural

antioxidants which prevent free radical oxidation of macromolecules in

cells and have special pharmacological activities (Walton and Brown,

1998; Quiroga et al., 2001).

Seeds are the source of several enzymes (Olczak et al., 1997;

Kaneko et al., 1990). During the germination of seeds, mostly

enzymes are activated and mobilize the store food reserves through

their catalytic activities. The acid phosphatase is one of these enzymes

which releases inorganic phosphate from reserved phosphate organic

compounds in response to phosphate deficiency (Duff et al., 1994),

salt stress (Pan, 1987) and water deficit (Barrett-Lennard et al.,

1982). In seeds and seedlings, increased acid phosphatase activity

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

213

either due to protein synthesis or by imbibition, catalyzes the

hydrolysis of many phosphate esters of sugars and phosphorylated

compounds to release inorganic phosphate for growth and

development (Akiyama and Suzuki, 1981; Hoehamer et al., 2005)

while in roots, increased secretion of extracellular acid phosphatases

act on insoluble organic phosphate compounds in soils to convert into

soluble inorganic phosphate to be taken up by plants (Panara et al.,

1990) in growing stage of seeds.

Acid phosphatases (EC 3.1.3.2) catalyzing the hydrolysis of

various phosphomonoesters at pH below 6.0, have been extensively

studied in leaves, roots and seeds (Tejera-Garcia et al., 2004).

Similarly, some studies on the purification and characterization of acid

phosphatase enzymes have also been done on the plant seedlings

(Gonnety et al., 2004; Tabaldi et al., 2008).

The present study deals with isolation of acid phosphatase

enzyme from seeds of weeds viz. A.fatua (wild oat), and Chenopodium

album (common lambsquarters) and its biochemical properties. In

addition to this, phytochemical constituents present in these seeds are

also investigated qualitatively and quantitatively.

MATERIALS AND METHODS

Seeds of wheat weeds A. fatua and C.album were obtained

from Ayub Agriculture Research Institute, Faisalabad Pakistan.

Enzyme Assays

The acid phosphatase activity was estimated according to the

method of Nadir et al. (2012). The reaction mixture consisted of 950µl

of 4mM substrate in 0.1M acetate buffer pH 5.5 and 50µl of the

enzyme. After incubation for 5 min at 37o C, the reaction was stopped

by the addition of 4 ml 0.1M KOH. The yellow color obtained, was

measured at 405 nm. One unit of enzyme was defined as the amount

of enzyme which catalyses the hydrolysis of 1 nano mole of substrate

p.nitrophenyl phosphate under above assay conditions. The molar

extinction coefficient of 17600 M-1cm-1 was used and specific activity as

units per mg of protein.

To determine pH optima, acid phosphatase activities were

assayed at various pH values from 4 to 6.2. The pH range was covered

with citrate buffer. Similarly, the activities were determined at

different temperatures ranging from 35 to 70o C to study optimum

temperature.

The pH stability of the enzyme was studied in 0.1M citrate

buffer pH range 4 to 6.2. After 1h pre-incubation at 37o C, aliquots

were taken and residual enzyme activities were assayed as described

above.

Asma Saeed et al., Some phytochemical and enzymatic ... 214

The kinetic constants such as Michaelis-Menten constant (Km),

maximal velocity constant (Vmax) and specificity constant (Vmax/ Km)

were determined at pH 5.6 at 37o C. The rate of hydrolysis of

p.nitrophenyl phosphate was measured under standard assay

procedure. These kinetic constants were estimated from Lineweaver-

Burk plot using substrate concentrations ranging from 0.05- 4 mM

p.nitrophenyl phosphate. In substrate specificity study, the activities

against other phosphorylated compounds were estimated by

determination of released inorganic phosphate according to Black and

Jones method (1983).

Thermal inactivation of each enzyme was determined at 37o C

and at its optimal temperature (55°C for C. album and 60°C for A.

factual). The separate enzyme solutions at pH 5.6 were pre-incubated

at each temperature for upto100 min. The small portions were taken

out at interval of 10 min and the remaining activity in each case was

assayed as usual. Thermal denaturation study was carried out by pre-

incubating each enzyme at temperatures from 35o C to 80o C. for about

10 min. The remaining enzyme activities were estimated as per

routine procedure described above.

Protein determination

Method of Lowry et al. (1951) was used to estimate protein

concentration. To 1ml of a protein solution, 0.5ml of 1N NaOH solution

was added and mixture was shaken with vortex mixer. After 30 min,

5ml of a mixture containing 100 volumes of 2% Na2CO3, 1 volume of

2% sodium tartrate and 1 volume of 1% CuSO4, was added. After 15

min 0.5 ml diluted Folin-Ciocalteu reagent (1/1, v: v) was added and

the content was mixed vigorously. After 30 min the absorption was

measured at 720 nm. The amount of protein was calculated from the

standard curve using bovine serum albumin as standard protein, while

protein concentrations of the column effluent fractions were estimated

by measuring their absorption at 280 nm.

Preparation of extracts and partial purifications

Acid phosphatases were purified from two sources by a

procedure that was slightly modified as described by Baig et al.

(1986). The seeds were crushed in powder form and the crude enzyme

was extracted by shaking the weed seed powder in 0.01 M Tris-HCl

buffer pH 7.0 (1 g powder/10 ml buffer) for 1 h. It was then

centrifuged at 4000-5000 rpm (Rotor JA-14) for 30 min. The clear

supernatant, thus obtained was used for partial purification of the

enzyme. The supernatant was dialysed against above buffer with

several times replacement of new buffer until the ionic strength of

dialysate was the same as that of buffer. The dialysate was centrifuged

at same speed to remove precipitated protein. The 25 ml of

supernatant sample was applied to DEAE-Cellulose column (2x20 cm).

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

215

The column was eluted buffer. Five ml/10 min each fraction was

collected. When all unbound protein was washed out from the column,

a linear sodium chloride salt gradient from 0 to 0.5 M (100 ml buffer

+100 ml NaCl in buffer) was applied with same speed. The fractions

representing the enzyme peak were pooled, concentrated to few ml by

ultrafiltration and applied on to Sephadex G-100 column (1.8x 60 cm).

The column was eluted with Tris buffer at flow rate 40 ml/h and 5 ml

fractions were collected. The active fractions were combined and

stored for further study.

Preparation of alcoholic extracts.

The seeds were ground to powder and suspended (20g) in 100

ml of 80 % methanol. It was left for one week to complete the

extraction process. Every day the mixture was vigorously mixed and

then it was filtered through Whatman filter paper No.1. The filtrate

was concentrated under vacuum distillation until dry mass is obtained.

To perform tests, 0.1 g dry mass was suspended in 10 ml of 10%

dimethyl suloxide.

Phytochemical screening

Following chemical tests were carried out for qualitative

determination of constituents on the methanolic extract using standard

procedures.

Tannins

A volume of 0.5 ml of extract was mixed with water and

heated. To it, few drops of 1% FeCl3 were added. The appearance of a

blue color indicated the tannins (Trease and Evans, 1989).

Saponins

Up to 0.5ml of extract was mixed with water and shaken

vigorously. Formation of persistant froth indicated the saponins

(Kumar et al., 2009).

Flavonoids

To 0.5ml of extracts, 1 ml of sodium hydroxide was added.

Yellow color was formed which becomes colorless on the addition of

dilute acid, indicated the presence of flavonoids. (Roopashree et al.,

2008).

Steriods

To 0.5ml of extract, few drops of H2SO4 were added followed by

adding 1ml of acetic anhydride. The appearance of blue-green

coloration indicated the presence of steroids (Kokate, 1994).

Terpenoids

To 0.5ml of extract, few drops of chloroform were added and

then conc.H₂SO₄ was run down the sides of test tube slowly so that

two layers are formed. The formation of a reddish brown ring at the

junction of the two layers indicated the presence of terpenoids (Evans,

1997).

Asma Saeed et al., Some phytochemical and enzymatic ... 216

Glycosides

To 0.5ml of extract containing glacial acetic acid, few drops of

FeCl3 were added and then conc.H₂SO4 was run down the sides of test

tube slowly. The appearance of a purple ring at the junction of the two

layers indicated the presence of glycosides (Sofowara, 1984).

Alkaloids

The extract was heated with 2% H2SO4. On cooling,

Dragendroff’s reagent was added orange red precipitation was

observed, showing the presence of alkaloids (Sofowora, 1982).

Quantitative determination of the chemical constituents

Quantitative determination of alkaloids, tannins and saponins

was done by using methods as described by Harborne (1973), Van-

Burden and Robinson (1981), Obadoni and Ochuko (2002),

respectively. For these tests, fat free seed powder was prepared. Two

g of seed powder were defatted with 100 ml of diethyl ether using a

soxhlet apparatus for 2 h. For flavonoids determination, a method of

Bohm and Kocipai-Abyazan (1994) was employed. Ten grams of the

seed sample was extracted with 100 ml of 80 % aqueous methanol

and solution was filtered through Whatman filter paper No 42. The

filtrate was evaporated to dryness and weighed to a constant weight.

RESULTS AND DISCUSSION

Acid phosphatases from two sources viz. A.fatua and C. album

seeds were partially purified by chromatography on DEAE-Cellulose

and Sephadex G-100 columns to make an appropriate comparison of

their properties and characteristics.

Purification was obtained from DEAE-Cellulose column. The

enzyme was completely bound to the top of the column while

unwanted proteins were washed out with equilibration buffer. The acid

phosphatase activity was eluted with linear gradient between 0.05M-

0.25 M NaCl in the same buffer. Two to three times purification of the

enzyme was achieved with 75-82 % yield with respect to starting

material loaded the column in all two cases. The active fractions of

anion exchange chromatography were subjected to Sephadex G-100

column chromatography. The acid phosphatase enzyme came out as

one peak. Ten to thirteen times more purification was achieved from

Sephadex G-100 chromatography. The specific activity was increased

from 46 to 460 U/mg in case of A.fatua while specific activity was

increased from 78.3 to 999 U/mg in case of C.album (Table-1). The

typical patterns of chromatography are shown in Fig. 1. The final

preparations had specific activity of 460-999 U/mg of protein with

approximately 23-37 fold purification (S.A of the final step/ S.A of the

initial step during purification) and had overall yields about 60 % (with

respect to starting materials).

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

217

Catalytic properties of the enzyme

At 37oC, acid phosphatases from both sources presented

stability over pH from 5 to 6 conserving at least more than 90 % of

total activities and seemed to be stable at 4o. No activity loss could be

detected even after incubation at 20o C for 24h. Both enzymes showed

pH optima of 5.6 (Fig. 2). The temperature optima of 55o C for

Chenopodium album seeds and 60o C for A.fatua seeds were obtained

(Fig. 3).The bell shaped curves were seen in all cases. The thermal

inactivation experiments indicated that at 37o C, the enzymes from

both sources were found stable for 100 min (Fig. 4) but at their

optimal temperatures, enzymes from C. album and A.fatua seeds were

less stable with half lives of 46 and 63 min, respectively. The thermal

denaturation showed that enzymes retained full activities at their

optimal temperatures. Above these optima, their enzymatic activities

decreased quickly as the temperatures increased (Fig.5). The enzymes

appeared to be completely denatured at 80o C. The kinetic parameters

of the two enzymes were studied using p.nitrophenyl phosphate

(Table-2). The Km values were found in range of 0.2-0.3mM. The

activity of acid phosphatases toward various substrates is shown in

Table-3.The p.nitrophenyl phosphate, phenyl phosphate, ATP and

sodium pyrophosphate were found good substrates. Among natural

substrates, ATP had high specificity. In general, these enzymes

hydrolyse large number of substrates and thus have broad specificity

for substrates. Kinetic parameters suggested that the enzyme from C.

album was more active than that of A.fatua.

The phytochemicals analyses of the plant seeds (Table-4)

showed that the seeds contained flavinoids, saponins, tennins,

steroids, glycosides, alkaloids and terpenoids. But quantitative

determinations of these constituents (see in Materials and Methods)

indicate that these were found rich in alkaloids, flavonoids and tannins

(Table-5). Both seeds contained highest percentage of tannins (16-

18%) followed by flavinoids (4-8%), alkaloids (2%) and saponins (less

than 1%). The flavonoids as an important class secondary metabolites

prevent free radical oxidation of molecules in the body (Cho et al.,

2004). So A. fatua and C. album may be used to prevent oxidative

stress. Flavoanoids also possess many other activities such as anti-

inflammatory and antiviral activities (Bbosa, 2010). Glycosides are

reported to decrease the blood pressure (Nyarko and Addy, 1990).

Phenolic compounds such as gallic, catechin have got much

importance for their significance antioxidant properties, prevent

oxidative damage and for the perfection of health toward various

diseases (Li et al., 2013). Suchandra et al. (2007) also reported the

phenolic compounds in M. fragrans which have great potential to

scavenge DPPH free radicals and inhibit lipid peroxidation. Tannins are

Asma Saeed et al., Some phytochemical and enzymatic ... 218

polycyclic aromatic compounds and constitute active component of

seeds cotyledon. These possess different anti-diarrheal activities

(Enzo, 2007). Saponins act as anti-nutrient in seeds possess

cholesterol lowering activity and deleterious properties (Akubugwo et

al., 2007). Terpenoids have pharmaceutical applications such as

artemisinin and taxol medicines are used for malaria (Goto et al.,

2010). Steroidal compounds have great importance as sex hormones

(Okwu, 2001). Similarly, alkaloids have got pharmacological

importance as enzyme inhibitors and growth terminators (Nazrullacv

et al., 2001).

CONCLUSION

For the first time, partial purification of acid phosphatases from

two sources, A. fatua and C. album seeds was reported. The enzymes

were purified to specific activity of 460-1000 U/mg of protein with 20-

40 fold purification and had overall recovery of about 60 %. Both

plants contained relatively high concentrations of tannins and

flavinoids while alkaloids and saponins were present in low

concentrations. The antioxidant activity may be expected due to the

presence of higher phenolic contents. Thus these plants may be used

to prevent oxidative stress and their bioactive phytochemical

constituents may be considered on medicinal merit.

ACKNOWLEDGEMENTS

The authors are indebted to Ayub Agriculture Research

Institute, Faisalabad Pakistan, for the gift of weed seeds.

Table-1. Purification steps of Avena fatua and Chenopodium album

acid phosphatases. Purification steps Avena

factua Chenopodiu

m album

S.A (U/mg)

P.F Act.Rec. (%)

S.A (U/mg)

P.F Act.Rec. (%)

Crude extract 20 1 100 27 1 100

DEAE-Cellulose chromatography

46 2.3 82 78.3 2.9 75

Sephadex G-100 chromatography

460 23 63 999 37 57

S.A. specific activity; P.F. purification factor; Act. Rec. recovery

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

219

Table-2. Kinetic parameters of enzymes from two sources towards

p.nitrophenyl phosphate

Avena fatua Chenopodium album

Km 0.23 mM 0.3 mM

Vmax 510 nmol.sec-1mg-1 1023nmol.sec-1mg-1

Vmax/ Km 2217 3410

Table-3. Substrate specificity of acid phosphatases from Avena fatua

and Chenopodium album

Substrates (% Activity)

Avena fatua

(% Activity) Chenopodium

album

pNPP 100 100

Phenyl phosphate 92 97

Glucose-1-phsphate 2 10

Glucose-6-phsphate 12 14

Fructose-1-phsphate 4 8

Fructose-6-phsphate 15 18

Sodium pyrophosphate 67 75

Adeosine tri phosphate 112 128

Sodium phytate 22 31

The enzyme activity was expressed as a percent of that of the same

enzyme towards p.nitrophenyl phosphate as 100

Table-4. Qualitative phytochemical constituents of weeds seeds

Weed

plants

Flavin

oids

Sapo

nins

Tanni

ns

Stero

ids

Glyco

sides

Alkaloi

ds

Terpe

noids

Avena

fatua

+ + + - + + +

Chenopodi

um album

+ + + - + + +

Table-5. Quantitative (percent) phytochemical constituents of seeds

of weed plants

Weed plants Alkaloids Saponins Flavinoids Tannins

Avena fatua 1.91 0.73 4.1 17.93

Chenopodium

album

2.23 0.83 8.7 16.14

Asma Saeed et al., Some phytochemical and enzymatic ... 220

Figure 1 (A). DEAE-Cellulose chromatography

25 ml sample was applied and fractions of 5 ml were collected

with flow rate of 30 ml/h. The arrow indicates the start of linear

gradient. (B). Sephadex G-100 chromatography.

The column was eluted with buffer at flow rate of 10 ml/15 min

and 5 ml fractions were collected. Ordinates: protein at 280 nm

(); acid phosphatase activity, ΔA405 () and NaCl linear

gradient (∆∆)

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

221

Figure 2 pH optima of acid phosphatases from A. factua and C.

album, A. factua (…●…); C. album ■

Figure 3. Temperature optima of acid phosphatases from A. factua

and C. album, A. ( …∆…); C. album

Asma Saeed et al., Some phytochemical and enzymatic ... 222

Figure 4. Thermal inactivation of acid phosphatases from A. fatua and

C. album. A. fatua ( ♦ C. album ■

A. fatua ▲ C. album ( ♦

Figure 5. Thermal denaturation of acid phosphatases from A. fatua

and C. album. A. fatua (…▲…); C. album

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

223

REFERENCES CITED

Akiyama, T. and H. Suzuki.1981. Localization of acid phosphatase in

aleurone layers of wheat seeds. Pfanzenphysiol. 101:131.

Akubugwo, I. E., A. N. Obasi and S. Ginika. 2007. Nutritional Potential

of Leaves and Seeds of Black Nightshade Solanum nigrum L.

var virginicum from Afikpo-Nigeria. Pakistan J. Nutr., 6(4):

323-326.

Abbas, M.N., S.A. Rana, M. Shahid, N. Rana, M. Mahmood-ul-Hassan

and M. Hussain. 2012. Chemical evaluation of weed seeds

mixed with wheat grains at harvest. J. Animal Plant Sci. 22(2):

283-288.

Bbosa, G. S. 2010. Medicinal Plants used by traditional Medicines

Practitioners for the treatment of HIV/AIDS and related

conditions in Uganda. J. Ethnopharmacol., 130(1): 43-53.

Baig, M.R., A. Saeed., Z.A.khan and I.U. Khan. 1986. Comparaive

study of acid phosphatases from maize, millet and sorghum

seeds. J. Chem. Soc. Pak., 8(2): 237-239.

Barrett-Lennard, E. G., A.D. Robson and H. Greenway. 1982. Effect of

phosphorus deficiency and water deficit on phosphatase

activities from wheat leaves. J. Exp. Bot., 33(4): 682-693.

Bibi, S., K. B. Marwat, G. Hassan, and N. M. Khan. 2008. Effect of

herbicides and wheat population on control of weeds in wheat.

Pak. J. Weed Sci. Res. 14(3-4): 111-119.

Black, M.J. and M.E. Jones. 1983. Inorganic phosphate determination

in the presence of a labile organic phosphate: assay for

carbonyl phosphatase activity. Anal. Biochem., 135(1): 233-

238.

Boham, B. A. and R. Kocipai-Abyazan.1994. Flavonoids and

condensed tannin from leaves of Hawaiian Vccinium

vaticulatum and V. calycinium. Pacific Sci., 48: 458-463.

Cheema, M. S., M. Akhtar, and M. S. Iqbal. 2006. Performance of

different herbicides in wheat under irrigated conditions of

southern Punjab, Pakistan. Pak. J. Weed Sci. Res. 12(1-2): 53-

59.

Cho, M. J., L. R. Howard., R. L. Prior, and J. R. Clark. 2004. Flavonoid

glycosides and antioxidant capacity of various blackberry and

red grape genotypes determined by high-performance liquid

chromatogrphy/mass spectrometry. J. Sci. Food. Agric.,

84(13): 1771-1782.

Duff, S. M.G., G. Sarath and W.C. Plaxton. 1994. The role of acid

phosphatase in plant phosphorus metabolism. Plant Physiol.

90(4): 791-800.

Enzo, A.P. 2007. Traditional plants and herbal remedies used in the

treatment of diarrheal disease: Mode of action, quality, efficacy

Asma Saeed et al., Some phytochemical and enzymatic ... 224

and safety considerations. Modern Phytomedicine Turning

Medicinal Plants into Drugs. Wiley VCH Verlag GmbH & Co.

KGaA, Weinheim. pp. 248-260.

Evans, W.C. 1997. Pharmacology. Harcourt Brace and Co., Singapore,

pp. 226-228.

Gonnety, J.T., S. Niamke., B.M. Faulet., E.J.N. Kouadio and L.P.

Kouame. 2006. Purification and characterization of three low

molecular weight acid phosphatase from peanut (Arachis

hypogaea) seedlings. Afr. J. Biotechnol., 5(1): 35-44.

Goto, T., N. Takahashi, S. Hirai and T. Kawada. 2010. Various

Terpenoids Derived from Herbal and Dietary Plants Function as

PPAR Modulators and Regulate Carbohydrate and Lipid

Metabolism. Hindawi Publishing Corporation PPAR Research.

Article ID 483958, p. 9.

Harborne, J. B. 1973. Phytochemical Methods, Chapman and Hall, Ltd.

London, pp. 49-188.

Hoehamer, C. F., C. S. Mazur and N. L. Wolfe. 2005. Purification and

partial characterization of an acid phosphatase from spirodela

oligorrhiza and its affinity for selected organophosphate pesticide.

J. Agric. Food Chem., 53(1): 90-97.

Hussain, F., M. Islam and A. Zaman. 2006. Ethnobotanical profile of

plants of ShawarValley, District Swat, Pakistan. Int. J. Biol.

Biotech., 3(2): 301-307.

Kaneko, J., M. Kuroiwa., K. Aoki., S. Okuda., Y. Kamio and K. Izaki.

1990. Purification and properties of acid phosphatase from

axes and cotyledons of germinating soybeans. Agric. Biol.

Chem., 54(3): 745-751.

Kokate, C. K. 1994. Practical pharmacognosy. 4th ed., Vallabh

Prakashan New Delhi. 1:15-30.

Kumar, A., R. Ilavarasan., T. Jayachandran., M. Decaraman., P.

Aravindhan., N. Padmanaban and M. R. V. Krishnan. 2009.

Phytochemical investigation on a tropical plant. Pak. J. Nutri.,

8(1): 83-85.

Li, G., X. Ge and R. Zhao. 2013. Characterization and Antioxidant

Activity of the Complex of Pomegranate Polyphenol Extract and

Lecithin. J. Chem. Soc. Pak., 35(3): 994-998.

Lowry, O.H., N.J. Rosebrough., A.L. Farr and R.J. Randal.1951.

Protien measurement with the Follin phenol reagent. J. Biol.

Chem., 193(1): 265-275.

Nadir, S., A.Saeed, R. Naz, A. Siddiqua, M. Sherazi, S.M. Wazir and A.

Saeed. 2012. Isolation, purification and characterization of acid

phosphatase from germinating Vigna radiata seeds. J. Chem.

Soc. Pak. 34(3): 717-727.

Pak. J. Weed Sci. Res., 22(2): 211-226, 2016

225

Nazrullaev, S. S., I. A. Bessonova and K. S. Akhmedkhodzhaeva.

2001. Estrogenic activity as a function of chemical structure in

Haplophyllum quinoline alkaloids. Chem. Nat. Compd., 37: 551-

555.

Nyarko, A.A and M.E. Addy. 1990. Effects of aqueous extract of

Adenia cissampeloides on blood pressure and serum analyte of

hypertensive patients. Phytother. Res., 4(1): 25-28.

Obadoni, B. O. and P. O. Ochuko. 2002. Phytochemical studies and

comparative efficacy of the crude extracts of some homostatic

plants in Edo and Delta States of Nigeria. Global J. Pure Appl.

Sci., 8 (2): 203-208.

Olczak, M., W. Watorek and B. Morawieka. 1997. Purification and

charactertization of acid phosphatase from yellow Lupin

(Lupinus luteus) seeds. Biochem. Biophys. Acta. 1341: 14-25.

Okwu, D.E. 2001. Evaluation of the chemical composition of

indigenous spices and flavouring agents. Global J. Pure Appl.

Sci., 7(3): 455-459.

Pan, S. 1987. Characterization of multiple acid phosphatase in salt-

stressed spinach leaves. Aust. J. Plant Physiol., 14: 117-124.

Panara, S., S. Pasqualini and M. Antonielli. 1990. Multiple forms of

barley root acid phosphatase: Purification and some

characterization of the major cytoplasmic isoenzyme. Biochem.

Biophys. Acta. 1037(1): 73-80.

Quiroga, E. N., A. R. Sampietro and M. A. Vattuone. 2001. Screening

anti-fungal activities of selected medicinal plants. J.

Ethnopharmacol., 74(1): 89-96.

Qureshi, R. and G. R. Bhatti. 2001. Determination of weed

communities in wheat field of district Sukhur. Pak. J. Bot.,

33(1): 109-115.

Roopashree, T. S., R. Dang, R. H. S. Rani and C. Narendra. 2008.

Antibacterial activity of antipsoriatic herbs: Cassia tora,

Momordica charantia and Calendula officinalis. Intl. J. App. Res.

Nat. Prod., 1(3): 20-28.

Siddiqui, I. and R. Bajwa. 2001. Variation in weed composition in

wheat fields of Lahore and Gujranwala divisions. Pak. J. Biol.

Sci., 4(Suppl.5): 492-504.

Sofowora, A. 1982. Medicinal Plants and Traditional Medicine in Africa.

John Wiley & Sons, New York, pp. 256.

Sofowara, A. 1984. Medical Plants and Traditional Medicines in Africa.

Spectrum Book Ltd, Ibadan, Nigeria. pp. 289.

Suchandra, C., C. Zareena, A. Gautam and S. Prasad. 2007.

Antioxidant activity of some phenolic constituents from green

pepper (Piper nigrum L.) and fresh nutmeg mace (Myristica

fragrans). Food Chem., 101(2): 515-523.

Asma Saeed et al., Some phytochemical and enzymatic ... 226

Tabaldi, L.A., R. Ruppenthal., L.B. Pereira., D. Cargnelutti, J.F.

Goncalves, V.M. Morsch and M.R.C. Schetinger. 2008.

Presence of multiple acid phosphatase activity in seedlings of

cucumber, raddish and rocket salad. Ciencia Rural. Santa Maria.

38(3): 650-657.

Tejera-Garcia, N.A., M. Olivera., C. Iribarne and C. Lluch. 2004.

Partial purification and characterization of a non-specific acid

phosphatase in leaves and root nodules of phaseolus vulgaris.

Plant Physiol. Biochem., 42: 585-591.

Trease, G. E. and M. D. Evans. 1989. A Textbook of Pharmacognosy.

13th ed. Builler Tindall and Caussel London. pp. 176-180.

Usman, K., S.K. Khalil, A.Z. Khan, I.H. Khalil, M.A. Khan and

Amanullah. 2010. Tillage and herbicides impact on weed control

and wheat yield under rice-wheat cropping system in

Northwestern Pakistan. Soil Tillage Res., 110(3): 101-107.

Van-Burden, T. P. and W.C. Robinson. 1981. Formation of complexes

between protein and tannin acid. J. Agric. Food Chem., 1(4):

77-82.

Walton, N.J. and D.E. Brown. 1998. Chemicals from Plants:

Perspectives on Plant Secondary Products; Imperial College

Press, London, UK.

Yadav, N., N. Vasudeva., S. Singh and S.K. Kumar. 2007. Medicinal

properties of genus Chenopodium Linn. Nat. Prod. Radiance.

6(2): 131-134.